! PROJECT SUMMARY/ABSTRACT After proteins are synthesized on the ribosome, their activity, stability, and localization are often regulated by post-translational modification (PTM) enzymes. Such modifications are essential for cellular function in all domains of life, and dysregulation of PTM processes is linked to myriad adverse medical conditions, including cancer and heart disease. A long-term goal of this work is to understand how PTM enzymes select their targets, particularly where recognition elements on the target protein are distant from actual sites of modification?a phenomenon termed ?alloselectivity.? A second long-term objective is to establish a general system for engineering proteins and peptides to be modified with user-defined PTMs. The proposed work uses the micrococcin biosynthetic pathway as a model analytical system. Micrococcin is an antimicrobial compound resulting from over 20 post-translational chemical conversions on a 14-amino acid precursor peptide substrate. The subjects of this study are the micrococcin biosynthetic proteins TclE, TclI, TclJ, and TclN. TclE is the substrate peptide, TclJ and TclN are modification enzymes, and TclI is the coupling protein that recruits TclJ/N to the TclE substrate. Proposed experiments probe the structural features underpinning this PTM complex, with an emphasis on the role of TclI as a crucial structural hub that physically joins the other three components. This four- protein complex can be functionally expressed at high levels in E. coli cells. The specific aims of the project are to (i) characterize substrate (TclE) recognition by TclI using a series of mutations that are guided by structural predictions, (ii) characterize the stable interactions between TclI and the TclJ/N enzymes, using a combination of mutational and analytical biochemical approaches, and (iii) design, build, and test prototypes of engineered PTM complexes that use elements of TclI and TclE to couple heterologous protein kinase domains to engineered peptide substrates. This work will be carried out primarily by an interdisciplinary team of undergraduate researchers. They will employ methods that include plasmid manipulation, site-directed mutagenesis, in vivo bacterial two-hybrid analyses, chromatographic co-purification, chemical cross-linking coupled with mass spectrometric analysis, and computational protein structural modelling. !